Molecular marker 27W1 for breeding high fecundity prawn population and its application

Abstract

This invention provides a molecular marker 27W1 for breeding high-fertility Litopenaeus vannamei populations and its applications. The nucleotide sequence of the molecular marker 27W1 is shown in SEQ ID No. 1, wherein the allele frequency of G at base 301 in high-fertility Litopenaeus vannamei populations is ≥98.33%. This invention also provides primers for the molecular marker 27W1, including amplification primers with nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, and extension primers with nucleotide sequences shown in SEQ ID No. 4. The high-fertility molecular marker 27W1 for Litopenaeus vannamei can be used for early breeding of Litopenaeus vannamei, regardless of growth stage, to rapidly select parent populations with excellent reproductive traits and promote the breeding process of new high-fertility Litopenaeus vannamei varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker-assisted breeding of aquatic animals, specifically involving the molecular marker 27W1 for breeding high-fertility shrimp populations and its application. Background Technology

[0002] Litopenaeus vannamei, also known as the Pacific white shrimp, belongs to the phylum Arthropoda, class Crustaeea, order Decapoda, family Penaeidae, and genus Penaeus. It is the most produced farmed shrimp species in my country and the world, and one of the highest-value aquaculture species. In 2020, my country's aquaculture production exceeded 1.8 million tons, with a value exceeding 70 billion yuan, accounting for approximately 40% of the world's total production. my country's annual demand for broodstock shrimp exceeds 1 million pairs, and the demand for seedlings exceeds 1.5 trillion. Litopenaeus vannamei is characterized by high spawning capacity and the ability to spawn multiple times. However, in seedling production, there is significant individual variation in the reproductive capacity of female shrimp. During a production cycle, some female shrimp never spawn, while others can spawn multiple times. Developing new varieties with high reproductive capacity (spawning and spawning volume) is of great significance for improving seedling production efficiency and saving production costs. Reproductive traits are of medium to low heritability, and traditional breeding methods have made slow progress. However, molecular marker-assisted breeding technology can effectively accelerate the breeding process.

[0003] As a third-generation molecular marker, single nucleotide polymorphism (SNP) markers are characterized by high abundance, high density, strong stability, and co-dominance, making them the most widely used molecular marker technology in economically important crustaceans such as shrimp and crab. Currently, there is limited development of SNP markers related to the reproduction of Litopenaeus vannamei, and the industry lacks markers applicable to marker-assisted breeding. Therefore, developing highly fertile molecular markers is of great significance for the breeding of new Litopenaeus vannamei varieties. Summary of the Invention

[0004] This invention provides a molecular marker, 27W1, for selecting high-fertility shrimp populations and its application. Molecular marker 27W1 can be used for screening high-fertility Litopenaeus vannamei populations with high identification efficiency and accuracy.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0006] This invention provides a molecular marker 27W1 for breeding high-fertility shrimp populations, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0007] Furthermore, in a highly fertile population of Litopenaeus vannamei, the 301st base G of the molecular marker 27W1 is a candidate 27W1_SNP site.

[0008] Furthermore, the allele frequency in the high-fertility population of Litopenaeus vannamei with the base G is >98%.

[0009] Preferably, the allele frequency in the high-fertility population of Litopenaeus vannamei with the base G is ≥98.33%.

[0010] The present invention also provides primers for the molecular marker 27W1, the primers comprising amplification primers with nucleotide sequences as shown in SEQ ID No. 2 and SEQ ID No. 3 and extension primers with nucleotide sequences as shown in SEQ ID No. 4.

[0011] The present invention also provides the application of the molecular marker 27W1 or primers of the molecular marker 27W1 in screening for highly fertile Litopenaeus vannamei populations.

[0012] Further, the specific steps for screening Litopenaeus vannamei populations with high reproductive capacity are as follows: extract DNA from test samples within the Litopenaeus vannamei population (family, strain, or geographical population) and use it as a template. Perform flight mass spectrometry typing using the primers. If the allele frequency of G at the 301st base of the molecular marker in the typing results is ≥98.33%, then select the Litopenaeus vannamei population as a parent for breeding Litopenaeus vannamei with high reproductive capacity.

[0013] Furthermore, the number of test samples of the Litopenaeus vannamei family or population is more than 30.

[0014] Furthermore, in the flight mass spectrometry typing, the PCR amplification conditions were as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 56℃ for 25 s, extension at 72℃ for 30 s, repeated for a total of 40 cycles; final extension at 72℃ for 4 min; and storage at 4℃.

[0015] Furthermore, in the flight mass spectrometry typing, the conditions for the extension reaction were: pre-denaturation at 94℃ for 30s; denaturation at 94℃ for 5s, annealing at 52℃ for 5s, extension at 80℃ for 5s, repeated for a total of 40 cycles; final extension at 72℃ for 3min; and storage at 4℃.

[0016] This invention also provides the application of the molecular marker 27W1 in the genetic diversity analysis, germplasm identification, and genetic map construction of Litopenaeus vannamei.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] 1. The high-fertility molecular marker 27W1 for Litopenaeus vannamei provided by this invention can be used for early breeding of Litopenaeus vannamei without being limited by the growth stage, and can be used to quickly breed parent populations with excellent reproductive traits, thereby promoting the breeding process of new high-fertility varieties of Litopenaeus vannamei.

[0019] 2. The molecular marker 27W1 provided by this invention is used to detect the reproductive traits of Litopenaeus vannamei. The method is accurate, reliable, and simple to operate. It can effectively and quickly screen out high-fertility populations, assist in early breeding, increase the utilization efficiency of superior Litopenaeus vannamei broodstock, improve seedling yield, and promote the healthy reproduction of Litopenaeus vannamei. It is of great significance to the healthy aquaculture and development of Litopenaeus vannamei and has broad application prospects. Attached Figure Description

[0020] Figure 1 This is the result of flight mass spectrometry typing verification in the validation population in this invention. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0022] The Litopenaeus vannamei used in this invention were all sourced from Bangpu Seed Industry Technology Co., Ltd., a cooperative base of the Yellow Sea Fisheries Research Institute of the Chinese Academy of Fishery Sciences. Healthy female broodstock shrimp aged 10 months from 57 families underwent unilateral eyestalk removal to promote synchronous ovarian maturation. 8–10 shrimp per family were used, totaling 604 shrimp, with a weight of 50 ± 5 g. The eyestalk-removed female shrimp were then placed in six 16m... 3 Large pond, density 6-7 fish / m³ 2 Male parent shrimp were from the same family line, 8-9 per family, totaling 500 shrimp, weighing 45±5g. Male shrimp were introduced into a 3m³ family. 3 Cement pool, density 2-3 fish / m³ 2 The broodstock shrimp were fortified with three meals of sandworms and two meals of squid daily, with the total daily feed amount reaching 20% ​​of the broodstock shrimp's total weight, divided into five feedings per day, ensuring that sandworms were visible 24 hours a day. During the fortification period, the water temperature was maintained at 28-29℃, salinity at 30-31‰, pH at 7.8-8.2, with continuous oxygenation and an 80% water exchange rate per day. After 20 days of fortification, female shrimp with fully developed, orange-red ovaries were selected and placed in the male shrimp pond for natural mating, avoiding inbreeding. The number of spawnings and the number of spawns per spawning for all female shrimp were recorded over 30 days. A female shrimp reproductive selection index was established, calculated using the following formula:

[0023] y i =0.5*(a elfi -μ elf )*σ elf -1 +0.5*(a aeni -μ aen)*σ aen -1 (1)

[0024] In the formula, y i It is the reproductive index of the i-th female shrimp; a elfi and a aeni These are the spawning frequency and spawning quantity of the i-th female shrimp, respectively; μ elf and μ aen It is the average of the spawning frequency and spawning quantity of all female shrimp; σ elf and σ aen It represents the standard error of the spawning frequency and spawning volume of all female shrimp.

[0025] The 30 female shrimp with the highest index were considered the high-fertility group, and the 30 female shrimp with the lowest index (never showing gonadal maturity) were considered the low-fertility group. 30 high-fertility and 30 low-fertility female shrimp were selected to form a population with differential fertility. Muscle tissue was dissected and stored in cryovials in liquid nitrogen.

[0026] Example 1

[0027] I. Screening of candidate molecular markers related to female shrimp fertility

[0028] 1. Sequencing data filtering and alignment

[0029] DNA extraction from Litopenaeus vannamei muscle was performed using the CTAB method. The CTAB method, or Cetyltrimethylammonium Bromide method, involved adding 1 ml of 1×CTAB to a 1.5 ml sterile enzyme centrifuge tube; adding approximately 20 mg of sample to the tube and homogenizing at 60 Hz for 4 min; incubating in a 65°C water bath for 60 min; centrifuging at 8000 xg for 5 min at room temperature; transferring 900 μL of the supernatant to a new 2 mL sterile enzyme centrifuge tube; adding 450 μL of chloroform to the supernatant; tightly capping the tube and inverting to mix the sample for 30 s, until the solution was completely emulsified and white; centrifuging at 13000 xg for 10 min at room temperature; and transferring 800 μL of the supernatant to a new 1.5 mL sterile enzyme-free centrifuge tube. Beckman Agencourt Ampoule XP was used. Incubate the beads at room temperature in the dark for 30 minutes. Add 0.6 times the volume of the supernatant to the beads and mix thoroughly. Gently pipette the mixture more than 10 times and let it stand at room temperature for 5 minutes. Place the tube on a magnetic rack for 5 minutes until the solution is clear. Carefully aspirate and discard the supernatant. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack and add 200 μL of freshly prepared 80% ethanol. Let it stand at room temperature for 30 seconds and discard the supernatant, being careful not to disturb the magnetic beads. Wash the beads once more with 80% ethanol. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack and let the beads dry at room temperature for 2–5 minutes. Remove the 1.5 mL centrifuge tube from the magnetic rack and add 50 μL of 10 mM Tris HCl elution buffer. Gently pipette the mixture and let it stand at room temperature for 5 minutes. Place the 1.5 mL centrifuge tube on the magnetic rack and let it stand at room temperature for 5 minutes until the solution is clear. Carefully aspirate about 50 μL of the supernatant and transfer it to a new sample storage tube to obtain the purified DNA. DNA purity and integrity were analyzed by agarose gel electrophoresis; DNA purity (OD260 / 280 ratio) was detected using Nanodrop; and DNA concentration was precisely quantified using Qubit.

[0030] Equal volumes of qualified DNA samples were mixed into two pools, named the high-fertility DNA pool (HE) and the low-fertility DNA pool (LE), respectively. The mixed DNA samples were randomly fragmented into 500 bp fragments using a Covaris S2 / E210 fragment shredder. The DNA fragments underwent end repair, polyA tailing, sequencing adapter addition, purification, and PCR amplification to complete the library preparation. The constructed libraries were sequenced using an Illumina HiSeq 2500 in PE150 mode. The obtained raw reads were filtered to obtain clean reads for subsequent analysis. The sequencing data results are shown in Table 1.

[0031] Table 1 Overview of Sequencing Data Quality Control

[0032]

[0033] The filtered valid data were aligned using the Burrows-Wheeler alignment tool (BWA) software, and the alignment results were then processed using SAMTOOLS software to remove PCR duplicates. The average sequencing depth was above 30X, and the data could be used for subsequent analysis.

[0034] 2. Screening for differentially expressed regions in the genome

[0035] A sliding window strategy was used to select different genomic regions: a window size of 50 kb and a sliding step size of 25 kb were used. For the selected specific genomic region, the fold change in nucleotide diversity between two segregating groups (π-Ratio, calculated as π high-fertility group / π low-fertility group) and the fixation index (Fst) were calculated. Combining the results of Fst and π-Ratio, significant regions were screened based on the inter-population difference P < 0.01, and the intersection was considered a reliable candidate interval.

[0036] 3. Marker detection and annotation

[0037] SNPs and InDels in candidate regions were detected using the Unified Genotyper module in the Genome Analysis Toolkit (GATK) software. The filtering parameters were set to: -Window 4, -filter"QD<2.0||FS>60.0||MQ<40.0", and -G_filter"GQ<20". Ultimately, a total of 64,046 SNP markers and 20,295 InDel markers were identified within the genomically differential regions of the reproductive capacity population.

[0038] 4. SNP frequency difference analysis

[0039] Calculate the SNP-index and InDel-index of each site in the two mixed pools, and calculate the frequency difference distribution of SNP and InDel, with the following direction: △(index) = index (high fertility trait) - index (low fertility trait).

[0040] 5. Mark and Filter

[0041] Candidate SNPs and InDel markers were screened, and sites with a Δindex close to 1 or close to -1 in both populations were selected as priority sites for further validation. The screening criteria are as follows:

[0042] (1) Based on the annotation information of SNP sites, and sorted from high to low according to │△index│, sites in synonymous, non-synonymous mutations or upstream and downstream regions are selected first.

[0043] (2) Based on the annotation information of the InDel site, and sorted from high to low according to │△index│, sites with more than 5 inserted or deleted bases are selected first.

[0044] Finally, markers with significant differences in the frequency of reproductive segregation were screened out, resulting in a total of 374 SNP markers and 26 InDel markers.

[0045] Table 2. Statistical results of SNP and InDel detection and annotation.

[0046]

[0047]

[0048] II. Validation of molecular markers related to fertility

[0049] Time-of-flight mass spectrometry was used to validate candidate molecular markers related to oviposition traits in both high- and low-fertility populations. The specific steps are as follows:

[0050] (1) PCR amplification: Primers are designed on the flanking sequences of the marker site to amplify the DNA sequence containing the detection target;

[0051] (2) Single-base extension: SNP sequence-specific extension primers are added to the PCR amplification product, and single-base extension is performed using iPLEX technology. For the detection target, different genotypes differ only in the terminal target base of the extension;

[0052] (3) Mass spectrometry detection: The product after single-base extension is purified and transferred to a SpectroCHIP chip for mass spectrometry detection. The DNA is positively charged by laser irradiation and flies in the detection vacuum tube. The flight speed is inversely proportional to the mass of each extension product. Finally, the base type is determined by the position of the peak, thereby realizing genotyping.

[0053] (4) Based on the mass spectrometry results, the mutation markers of each individual were counted, and SPSS software was used to analyze whether the markers were related to the reproductive traits.

[0054] The specific steps are as follows:

[0055] 1. Primer design

[0056] Primer design software: Sequenom Genotyping Tools and Massarray Assay Design software were used to design PCR amplification primers and single-base extension primers for the target sites.

[0057] 2. PCR amplification

[0058] The PCR system in this invention is as follows: 1 μl template (10-30 ng / μl), 0.25 μl forward primer (10 μM), 0.25 μl reverse primer (10 μM), 0.5 μl buffer, 0.1 μl dNTPs (25 mM), 0.4 μl MgCl2 (25 mM), 0.1 μl HotStar Taq (5 U / μl), and 2.4 μl ddH2O.

[0059] After adding the sample according to the above system, PCR amplification was performed under the following reaction conditions: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 56℃ for 25 s, extension at 72℃ for 30 s, for a total of 40 cycles; final extension at 72℃ for 4 min; storage at 4℃.

[0060] 3. Alkaline phosphatase treatment of PCR products

[0061] (1) After the PCR reaction, the PCR product was treated with SAP (shrimp alkaline phosphtase) to remove dNTPs from the reaction. The SAP reaction solution formula is as follows (taking a single sample as an example): SAP Buffer 0.17 μl, SAP Enzyme (1.7 U / μl) 0.3 μl, ddH2O 1.53 μl.

[0062] (2) Add SAP reaction solution to PCR reaction plate using a pipette, 2 μl per well, seal and centrifuge.

[0063] (3) Place the PCR reaction plate containing the SAP reaction solution into the PCR instrument and run the following reaction program: 37℃, 40min; 85℃, 5min; store at 4℃.

[0064] (4) After the reaction is complete, remove the PCR reaction plate and centrifuge it briefly for later use.

[0065] 4. Extension reaction

[0066] (1) Prepare iPlex reaction reagents (taking a single sample as an example): iPlex Buffer (10X) 0.12μl, iPlex Termination mix (10X) 0.2μl, iPlex enzyme (2U / μl) 0.041μl, SAP Enzyme (1.7U / μl) 0.3μl, primer working solution 10μm 0.804μl, ddH2O 0.755μl;

[0067] (2) Use a pipette to add 2 μl of iPlex reaction solution to the PCR reaction plate, add 2 μl to each well, seal the plate and centrifuge;

[0068] (3) Place the PCR reaction plate into the PCR instrument and perform PCR amplification according to the following reaction conditions: pre-denaturation 94℃ for 30s; denaturation 94℃ for 5s, annealing 52℃ for 5s, extension 80℃ for 5s, repeat for a total of 40 cycles; final extension 72℃ for 3min; store at 4℃.

[0069] 5. Product purification

[0070] (1) Cover the resin evenly on the resin scraper and let it stand for 20 minutes.

[0071] (2) Centrifuge the PCR reaction plate at 1000 rpm for 1 min after the reaction is complete, add 25 μl of deionized water to each well, invert it on the resin plate (make sure it is fixed and does not move), then invert it and place the resin plate on the PCR reaction plate, tap it to make the resin fall into the PCR reaction plate, and seal it.

[0072] (3) Using the long axis of the PCR reaction plate as the center, flip the PCR reaction plate for 20 min, centrifuge at 3500 rpm for 5 min and set aside.

[0073] 6. Mass spectrometry detection

[0074] (1) Spotting the sample onto the Nanodispenser SpectroCHIP chip: Transfer the detection sample from the PCR reaction plate to the MassARRAY SpectroCHIP chip with a matrix covering its surface.

[0075] (2) Mass spectrometry detection using the MassARRAY Analyzer Compac;

[0076] (3) TYPER software was used to analyze the experimental results and obtain the typing data.

[0077] 7. Statistical Analysis

[0078] This invention screened and obtained a molecular marker 27W1, the nucleotide sequence of which is shown in SEQ ID No. 1. According to Table 3 and... Figure 1As shown, the allele frequency of G at position 301 in 27W1 is 98.33% in the high fertility group, while it is 83.33% in the low fertility group. The proportion of G at this position shows a significant difference between the fertility segregating populations (P<0.05). Therefore, a high fertility population can be considered to have an allele frequency of G ≥98.33% at this locus. The amplification primers for the molecular marker 27W1 are shown in SEQ ID No. 2 and SEQ ID No. 3 (Table 4), and the extension primers are shown in SEQ ID No. 4 (Table 4).

[0079] Table 3. Genotyping results of the 27W1 molecular marker

[0080]

[0081] Table 4 Primers for Molecular Markers

[0082]

[0083] The molecular marker 27W1 obtained in this invention can be used to assist in the selection of high-fertility populations (families, strains, or geographical populations). The specific application steps are as follows: DNA is extracted from at least 30 test samples within a Litopenaeus vannamei population and used as a template. Amplification primers 27W1-F and 27W1-R and extension primer 27W1-E for genotyping are performed using mass spectrometry. If the allele frequency of the 27W1 locus at base G (position 301) within the population is ≥98.33% in the genotyping results, then this population can be selected as a parent stock for breeding high-fertility Litopenaeus vannamei. In addition, the molecular marker 27W1 can also be used to analyze the transmissibility of Litopenaeus vannamei, identify germplasm, and construct a genetic map of Litopenaeus vannamei.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention. sequence list <110> Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences <120> Molecular marker 27W1 for breeding high-fertility shrimp populations and its application <150> 2022102023356 <151> 2022-03-02 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 601 <212> DNA <213> Artificial Sequence <400> 1 atatatatat atatgtatat gtgtggtgtg tgtgtgtgtg tgtgtgtgtg gtgtgtgtgt 60 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtttag agagagcg agcgagagcg 120 180 ggaggaaggg ggtagggagg gagagattaa gagagatagc gaggaggag gggaagagg 240 ggagggagat ggcagaagag gcagagggag aaaggaaaag ggcggtgagt cgggggagg 300 gggtaaggca gggaaggggc ggggttactt ctgccccatt cccatgtttc atatttgtca 360 taaaagttt cttcctgtaa gatttttgaa ggcatgttcc tctgccctct ttctcacatt 420 tcaaaggttt ctgtctggct ctgtcctttc gtgttgtccg ttggtgccct gcgtttgaaa 480 tgcaaaagga aaacataatg gatatccctt aataaatatg ttagtcatta tatatatata 540 600 g 601 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <400> 2 acgttggatg atgaaacatg ggaatggggc 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <400> 3 acgttggatg agagggagaa aggaaaaggg 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <400> 4 ggggccccgc cccttccctg ccttacc 27

Claims

1. A molecular marker 27W1 for selecting a high-fecundity Litopenaeus vannamei population, characterized in that, The nucleotide sequence of the molecular marker 27W1 is shown as SEQ ID No. 1; in the high-fecundity population of Litopenaeus vannamei, the base G at position 301 of the molecular marker 27W1 is a candidate 27W1_SNP site, and the allelic frequency of the base G in the high-fecundity population of Litopenaeus vannamei is >98%.

2. The primer of the molecular marker 27W1 according to claim 1, characterized in that, The primer comprises an amplification primer with the nucleotide sequence shown as SEQ ID No. 2 and SEQ ID No. 3, and an extension primer with the nucleotide sequence shown as SEQ ID No.

4.

3. Use of the primer of the molecular marker 27W1 according to claim 2 for screening Litopenaeus vannamei populations with high fecundity, characterized in that, The specific steps for screening the high-fecundity population of Litopenaeus vannamei are as follows: extracting the DNA of a test sample in a population of Litopenaeus vannamei, taking the DNA as a template, and performing MALDI-TOF MS typing by using the primer; if the allelic frequency of the base G at position 301 of the molecular marker in the typing result is >98%, the population of Litopenaeus vannamei is selected as a parent for breeding high-fecundity Litopenaeus vannamei.

4. Use according to claim 3, characterized in that, The number of test samples of the population of Litopenaeus vannamei is >30.

5. Use according to claim 3, characterized in that, In the MALDI-TOF MS typing, the PCR amplification conditions are as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 25 s, extension at 72°C for 30 s, and repeating for 40 cycles; final extension at 72°C for 4 min; and preservation at 4°C.

6. Use according to claim 3, characterized in that, In the MALDI-TOF MS typing, the extension reaction conditions are as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 52°C for 5 s, extension at 80°C for 5 s, and repeating for 40 cycles; final extension at 72°C for 3 min; and preservation at 4°C. In the MALDI-TOF MS typing, the extension reaction conditions are as follows: pre-denaturation at 94°C for 30 s; denaturation at 94°C for 5 s, annealing at 52°C for 5 s, extension at 80°C for 5 s, and repeating for 40 cycles; final extension at 72°C for 3 min; and preservation at 4°C.

Images